Sybr green qpcr master mix rox

Quantitative real‐time PCR was performed using the Arraystar SYBR® Green qPCR Master Mix (ROX+) (Arraystar, USA) on an Applied Biosystems 7500 FAST Real‐time PCR system (Applied Biosystems, USA). Relative quantification of target gene expression was calculated with 2^−ΔΔCT method by dividing the target lncRNA/reference gene ratio of a tested plasma sample by the target lncRNA/reference gene ratio of a reference sample. For the small sample size validation in the discovery set, we used a pooled sample from the ten GC subjects as the reference sample. In the large sample size validation, the RNA extracted from the human gastric cell line MGC‐803 was used as the reference sample on every PCR detection plate with all samples assayed in duplicate. The MGC‐803 cell line was gifted from the Laboratory of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute. The samples with the Ct value for a candidate lncRNA <36 are considered positive expression. All the primers for candidate lncRNAs or reference genes were synthesized by Augct Biotech (Beijing, China), and the sequences are shown in Table S1.

Three pair-matched surgically resected tissues were used to obtain total RNA. OD260/280 measurements on the NanoDrop® ND-1000 (NanoDrop, US) were used to evaluate the purity and concentration of RNA. Denaturing agarose gel electrophoresis was used to test RNA integrity. First-strand cDNA was synthesized using the rtStar™ First-Strand Synthesis Kit (Cat# AS-FS-001, Arraystar, US), cDNA was mixed with Arraystar SYBR® Green qPCR Master Mix (ROX+) (AS-MR-006-5, Arraystar), and snoRNA profiling was performed by using nrStar™ Human snoRNA PCR array according to the manufacturer's instructions (Arraystar, Inc, US) on ABI 7900 real-time PCR system (Applied Biosystems, Foster City, CA). The PCR array consisted of 384 primer sets for analyzing small nucleolar RNAs.

In this study, a commercial tRF and tiRNA PCR array from ArrayStar was used to examine 88 tRFs known to be amplified by PCR. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the cells. The concentration of the RNA was measured using a NanoDrop ND-1000 (Thermo Fisher Scientific, USA). First, DNase treatment and RNA cleanup were performed using a RNeasy^® MinEluteTM cleanup kit (Qiagen, Germany). The rtStarTM tRF&tiRNA pretreatment kit (Cat# AS-FS-005, Arraystar, USA) and rtStarTM First-Strand cDNA Synthesis kit (Cat# AS-FS-003, Arraystar, USA) were applied separately to pretreat tRF and tiRNA and synthetize first-strand cDNA with tRF-specific RT primers. The process included the following steps: 3’-terminal deacylation, 3’-cP removal and 5’-P addition, demethylation, 3’ adaptor ligation, reverse transcription primer hybridization, 5’ adaptor ligation and reverse transcription. Arraystar SYBR^® Green qPCR Master Mix (ROX+) (AS-MR-006-5, Arraystar, USA) was used to perform qRT-PCR, amplifying and quantifying the cDNA according to the manufacturer’s protocols. The expression levels of tRFs were standardized to that of 5S RNA, and the relative expression levels of genes were quantified by the 2^-ΔΔCT method.